Presenilin (PS) represents a family of multi-pass transmembrane proteins consisting of, among others, Sel-12 and Hop-1 in C. elegans and PS1 and PS2 in mammals. Presenilin is required for the proteolytic activation of Notch and the enzymatic processing of beta-amyloid precursor protein (APP), molecules that play pivotal roles in cell fate determination during development and Alzheimer's disease (AD) pathogenesis through aging respectively. Presenilin-dependent Notch processing and signaling is highly conserved and exhibit strong amino acid sequence preference. In contrary, cleavage of APP by presenilin occurs at multiple sites, generating two major beta- amyloid peptides: Abeta40 and Abeta42. Mutations in human PS cause early onset of familial Alzheimer's disease (FAD) and the pathogenic effect is most likely mediated by their ability to increase the ratio of Abeta42/Abeta40. However, it has been difficult to understand this apparent "gain-of-misfunction" mechanism because the FAD mutations spread throughout the entire molecule and the mutant protein is only partially active compared to wild-type presenilin in C. elegans. The current application is aimed at resolving these unsettling and sometimes conflicting data. We hypothesize here that PS FAD mutation leads to concurrent partial loss of function on Notch and APP. Reduced activity at the preferred Abeta40 site by the PS FAD mutation drives the increase in the Abeta42/Abeta40 ratio. This hypothesis allows interpretation of presenilin function and the effect of PS FAD mutation on Notch and APP pathways across all species under one mechanism. Furthermore, since PS-mediated Notch activity is highly conserved; we propose that the C. elegans presenilin represents a partial active allele in mammalian system exhibiting similar effects as the PS FAD mutant protein. We plan to test the hypothesis in vivo using two powerful transgenic mouse systems: One is the PS1 FAD knock-in system that allows determination of FAD mutation under physiological context and the other is our novel PS1 "rescue" system that makes it possible to test various presenilin alleles for their Notch developmental and AD pathogenic activities. Because of the indispensable role of presenilin in multiple biological processes and the robust pathogenic impact of the PS mutations in Alzheimer's disease pathogenesis, determining the molecular mechanisms underlying these presenilin-mediated pathways and the effect by the FAD mutations in vivo is crucially important. The studies proposed will lead to better evaluation of presenilin-based therapy as well as advanced understanding of AD pathogenesis in general.